Mycobacterium tuberculosis (Mtb) transmission.Antimicrobial resistance develops following the accrual of mutations in the bacterial genome, and may variably impact organism fitness and hence, transmission risk. Classical representation of tuberculosis (TB) dynamics using a single or two strain (DS/MDR-TB) model typically does not capture elements of this important aspect of TB epidemiology. To understand and estimate the likelihood of resistance spreading in high drug-resistant TB incidence settings, we used epidemiological data to develop a mathematical model of Mtb transmission model was developed to explore the progression from DS- to MDR-TB in The Philippines and Viet Nam. The models were calibrated using data from national tuberculosis prevalence (NTP) surveys and drug resistance surveys (DRS). An adaptive Metropolis algorithm was used to estimate the risks of drug resistance amplification among unsuccessfully treated individuals.A four-strain (drug-susceptible (DS), isoniazid mono-resistant (INH-R), rifampicin mono-resistant (RIF-R) and multidrug-resistant (MDR)) compartmental deterministic The estimated proportion of INH-R amplification among failing treatments was 0.84 (95% CI 0.79–0.89) for The Philippines and 0.77 (95% CI 0.71–0.84) for Viet Nam. The proportion of RIF-R amplification among failing treatments was 0.05 (95% CI 0.04–0.07) for The Philippines and 0.011 (95% CI 0.010–0.012) for Viet Nam.The risk of resistance amplification due to treatment failure for INH was dramatically higher than RIF. We observed RIF-R strains were more likely to be transmitted than acquired through amplification, while both mechanisms of acquisition were important contributors in the case of INH-R. These findings highlight the complexity of drug resistance dynamics in high-incidence settings, and emphasize the importance of prioritizing testing algorithms which allow for early detection of INH-R.The online version contains supplementary material available at 10.1186/s12879-022-07067-1. Despite being both a preventable and curable disease, more than 10 million people develop tuberculosis (TB) each year, with 1.4 million deaths in 2019 . In all Mathematical modeling is increasingly used to support programmatic optimization for TB –6. AccouModeling also offers an opportunity to quantify amplification and transmission of drug-resistant TB, by fitting dynamic models to observed data. We therefore aimed to incorporate epidemiological data into an empirically calibrated model, in order to explore parameter estimation for drug resistance amplification and transmission associated with both INH and RIF.Mtb transmission to capture five mutually exclusive health states with regards to TB infection and disease—susceptible (S), early latent (LA), late latent (LB), infectious (I) and recovered . The model included four TB strains: drug-susceptible , isoniazid mono-resistant , rifampicin mono-resistant  and MDR-TB (compartment subscript M). It is to be noted that the strains are not phylogenetically related.We designed a deterministic compartmental model of i), as well as a universal population-wide death rate (μ).We assumed homogenous mixing in a closed population:Mtb, they transition from the susceptible compartment (S) to the early latent compartment (LA). The force of infection (λ) associated with each strain  can either progress (within two-years) directly to the active disease compartment (I) at rate ε, or transition to the late latent compartment (LB) at rate κ. Progression from LB to the active disease state occurs at a much slower rate (ν), and is referred to as reactivation. Once individuals have entered the infectious compartment, one of the following six processes can occur: (1) the person may be correctly identified as having active DS-TB and commenced on treatment (rate τ), thence progressing towards cure and transitioning to the recovered (R) compartment; (2) person may be correctly identified to have DS-TB or DR-TB and commenced on treatment but experiences treatment failure without experiencing resistance amplification to other drugs and stay in the same infectious compartment; (3) spontaneous recovery (rate γ) with transition to the recovered compartment (R); (4) TB-related death (μi) (5) dying of natural causes or (6) the infecting strain could acquire resistance (αH and/or αR) to isoniazid (INH-R), rifampicin (RIF-R) or MDR-TB and move to IH, IR and ultimately to IM compartments. To capture the progressive accrual of resistance with each transition, only one level of additional resistance not already present can be obtained during a disease episode. People who have spontaneously recovered from past TB or successfully completed treatment are both represented as a single compartment (R) on the assumption that prognosis is equivalent regardless of the infecting strain from which each person recovers. Once treatment is complete, the recovered person can transition back to LA through reinfection, represented as δ. We define δ  to the recovered compartment (R). The transition from the infectious to the recovered compartment is represented using the parameter “τ”. τ is dependent on the TB detection rate “d” and the TSR and is mathematically expressed in Eq. People diagnosed with active TB are commenced on treatment upon identification and move from the infectious compartments  surveys  and drug-resistance prevalence from national DR-TB surveys . The detailed estimates are presented in Table We used WHO-reported TB notifications as a calibration target for both models. For Viet Nam, in 2018, 102,171 cases were notified and for The Philippines 382,543 cases were notified and we calibrated to the per capita notification rates corresponding to these values.An Adaptive Metropolis (AM) algorithm was used to estimate model parameters , includiThe AM algorithm  was usedFigure Table We observed higher proportions of drug resistance amplification for INH compared to RIF for both the high DR-TB incidence settings we simulated Fig. . The estThe model was used to estimate the proportions of incident DR-TB due to transmission compared to DR amplification Table . In the In The Philippines, the model estimates for amplification from DS to INH-R was 26 per 100,000 (95% CI 15–33) people, followed by 10 per 100,000 (95% CI 6–14) people then gain resistance to RIF and moving to the MDR compartment. Comparing this to acquiring RIF resistance first, we see 2 per 100,000 (95% CI 1–3) moving from DS to RIF-R, followed by only 0.05 (95% CI 0.02–0.08) people gaining resistance to INH to move to the MDR compartment. A similar observation was seen for Viet Nam, the model estimates for amplification from DS to INH-R shows 6 per 100,000 (95% CI 4–8) people followed by people 4 per 100,000 (95% CI 3–5) gaining resistance to RIF and moving to the MDR compartment. In case of DS to RIF-R transition the estimates were 0.08 per 100,000 (0.06–0.11) people, followed by 0.0007 per 100,000 (0.0004–0.001) people gaining resistance to INH to move to the MDR compartment.The posterior estimates of relative fitness associated with INH-R strains for The Philippines was 0.87 (95% CI 0.83–0.92) and 0.98 (95% CI 0.95–1.00) for Viet Nam. The relative fitness associated with RIF-R strains for The Philippines was 0.78 (95% CI 0.74–0.84) and 0.77 (95% CI 0.73–0.81) for Viet Nam. The relative fitness associated with MDR-TB strains in The Philippines was 0.67 (95% CI 0.58–0.71) compared to 0.64 (95% CI 0.56–0.75) for Viet Nam.Our study also provided information on estimates of CDR with high precision for both the settings, as inclusion of notification and prevalence of infection data for the analysis helped in constraining the parameter. The estimates obtained for The Philippines was 0.49 (95% CI 0.47–0.51) and for Viet Nam was 0.66 (95% CI 0.63–0.69).From this modeling study we were able to construct a model which successfully replicated epidemiological dynamics in two high burden TB settings, incorporating parameters drawn from microbiological fitness data. Using this model to explore the development of drug resistance in these contexts, we found that a much higher proportion of treatment failure resulted in amplification for INH-R rather than for RIF-R. This finding is consistent with observed higher rates of INH-R globally , 32 and One factor likely to play a significant role in preferential INH-R amplification is current methods of DR-TB detection which prioritize RIF’s resistance identification. According to WHO and many country guidelines, TB patients with strains found to be resistant to RIF need to start on a recommended MDR-TB treatment regimen. Longer MDR-TB regimens, and historical second-line therapy regimens, frequently have INH included in them, irrespective of resistance to INH being either undetermined or confirmed. Re-treatment regimens in particular have often incorporated prolonged durations of INH therapy—for example the category II regimen used in the Philippines comprised of 8 months of INH, RIF and ethambutol supplemented by streptomycin for the initial 2 months, and pyrazinamide for the initial 3 months (2SHRZE/HRZE/ 5HRE) , and oldS, as compared to starting from IR; the same applies for RIF-R amplification. We even assumed the fitness cost of MDR-TB is independent of that of INH-R of RIF-R. Therefore, these limitations can potentially influence the estimated risk of resistance amplification.In addition to programmatic insights, our model provides novel information on parametrizing CDR. This is important, as this parameter cannot be measured directly yet plays a significant role in informing robust mathematical model of TB transmission. As with any mathematical representation our model has certain limitations. Our model was calibrated to TB prevalence and DR surveys to estimate the risk of resistance amplification. But the definition of a TB case may change between surveys, even within the same country. We have adopted a simplified model structure that does not capture factors such as age, comorbidities and other heterogeneity associated with TB epidemics. These factors may affect the risk of resistance amplification. Our model is primarily built for pulmonary tuberculosis and does not include extra-pulmonary TB data, as our primary focus was on transmission. For the same reason, this model has been parametrized from adult TB data given the limited TB transmission from young children to others. In our model we assumed the risk of INH-R amplification is the same starting from IFurthermore, it is difficult to compare the results generated by our model with existing studies because the findings are novel and not many researchers have tried to estimate risk of monoresistance amplification of INH using a four strain TB transmission model. Two studies which reported similar findings were—(1) study conducted in the KwaZulu-Natal Province of South Africa for extremely drug resistant TB transmission, reported 84% of the participants failed treatment . (2) A splus [Historically, diagnosis of MDR-TB has been reliant on culture-based phenotypic testing, which in high-burden settings may be applied selectively, such as after treatment failure. As part of the global policy to control DR-TB, many high burden settings have pledged to deploy the molecular diagnostic assay Xpert MTB/RIF (detects resistance only in RIF), which is a nucleic acid amplification test that can be directly applied to sputum samples , 43. As plus , Abbott plus , BD MAX plus , Roche cplus  and wholplus . Furtherplus .An interesting observation was made between the estimates of the fitness cost of each resistant strain and their respective transmission dynamics for both the countries. We see that the RIF-R and MDR-TB have similar fitness in both countries indicating homogeneity of the population, while INH-R strains seem to have a considerable difference between the two countries. This is reflected in the transmission dynamics observed for the INH-R strains, where Viet Nam has a higher transmission rate compared to The Philippines. This could mean the INH-R strains in Viet Nam, although under significant drug pressure in the host, are more transmissible, perhaps due to strain-specific factors or local host/pathogen interaction. The same correlation cannot be made for RIF-R strains. They have similar fitness cost in both countries, but the transmission rate is higher in Viet Nam compared to The Philippines.H) for both the high burden DR-TB setting, we see that the fitness cost is inversely related to ρH. While INH-R strains in Viet Nam have a higher fitness cost than The Philippines, the number of individuals acquiring resistance due to treatment failure is lower in Viet Nam. The estimates for CDR were also higher in Viet Nam compared to The Philippines. As, ρH and CDR is directly proportional to the amplification rate as defined in Eq. H is inversely proportional to the fitness cost of INH-R strains. Therefore, an increase in the mycobacterial fitness for INH-R can lead to a potential increase in transmission rate but a decrease in the amplification rate. In case of RIF-R strains, the fitness cost was similar for both the settings. The slight difference in the amplification rate can be explained by the higher CDR observed in Viet Nam. Factors such as age, behavior, and gender ratio of the overall population, could possibly be a reason to see this type of differences. But it is beyond the scope of this paper to predict the effects of these external influences on this model.Comparing fitness cost of INH-R strains to the proportions of individuals who develop INH-R due to treatment failure (ρWhile rapid molecular diagnostics will continue to be important for programmatic adoption, it is also important to recognize that the principle of unrecognized resistance amplification demonstrated here can be repeated for any resistance not routinely addressed in diagnostic algorithms. It is therefore essential to incorporate genome sequencing into surveillance programs, to maximize the clinical and public health benefits . With reAdditional file 1: Figure S1. A detailed representation of the Mtb transmission model


A5) was synthesized. Thiscopolymer formed highly water-dispersed nanoparticles and aggregatedinto a mixed ion–electron conducting hydrogel when injectedinto a tissue model. First, we synthetically repeated most of thepublished methods for PEDOT-S at the lab scale. Surprisingly, analysisusing high-resolution matrix-assisted laser desorption ionization-massspectroscopy showed that almost all the methods generated PEDOT-Sderivatives with the same polymer lengths ; thus, the polymer length cannot accountfor the differences in the conductivities reported earlier. The maindifference, however, was that some methods generated an unintentionalcopolymer P(EDOT-S/EDOT-OH) that is more prone to aggregate and displayhigher conductivities in general than the PEDOT-S homopolymer. Basedon this, we synthesized the PEDOT-S derivative A5, thatdisplayed the highest film conductivity (33 S cm–1) among all PEDOT-S derivatives synthesized. Injecting A5 nanoparticles into the agarose gel cast with a physiological buffergenerated a stable and highly conductive hydrogel (1–5 S cm–1), where no conductive structures were seen in agarosewith the other PEDOT-S derivatives. Furthermore, the ion-treated A5 hydrogel remained stable and maintained initial conductivitiesfor 7 months (the longest period tested) in pure water, and A5 mixed with Fe3O4 nanoparticles generateda magnetoconductive relay device in water. Thus, we have successfullysynthesized a water-processable, syringe-injectable, and self-dopedPEDOT-S polymer capable of forming a conductive hydrogel in tissuemimics, thereby paving a way for future applications within in vivoelectronics.Injectable bioelectronicscould become an alternative or a complementto traditional drug treatments. To this end, a new self-doped p-typeconducting PEDOT-S copolymer ( A recent study suggested that,in order to avoid blood vessel rupture and bleeding in the brain,a syringe needle with diameter below 25 μm should be used forimplantation of bioelectronics.6 To meetthis goal, we envisioned a strategy that uses highly soluble nanoparticlesthat self-organize into a conducting structure when injected intoa tissue, by using a capillary with a diameter of about 25 μm.Injectable conductive hydrogels have shown great biocompatibilitybecause of their resemblance to body tissue. Conductive hydrogelsare in general composed of a hydrogel matrix  supplemented by a conductive polymer  and have conductivities in a lowmS cm–1 range.8 In the search for amaterial that allows for the installation of different functionalgroups to diversify functionality toward controlling solubility–aggregationproperties and biocompatibility, the polymer polydoped with poly(styrene sulfonate) (PEDOT:PSS)11 was of interest.PEDOT:PSS displays high conductivity and the possibility for structuralmanipulation of the EDOT monomer.16 Recently, a mixture of PEDOT:PSS and 4-dodecylbenzenesulfonicacid (DBSA) was reported to form hydrogels at room temperature after2–200 min, depending on the concentration of DBSA. This hydrogelformation was demonstrated by extruding a hydrogel with a syringeon a >400 μm diameter scale, and it showed a conductivityofabout 10–1 S cm–1, which is atthe higher end of hydrogel conductivities17 and higher than most tissues (<10–2 S cm–1).18 However, the hydrophilictwo-component pristine PEDOT:PSS can disintegrate and lose functionwhen exposed to excessive humidity or water because of the highlywater-soluble PSS component.11 To reducethe number of crucial components for function and the chemical bulk,we sought to remove the nonconductive bystander materials in the injectablesolution, that is, PSS and DBSA. An interesting solution to this isthe self-doped polythiophenes, for example, PEDOT derivatives witha covalently attached sulfonate group known as PEDOT-S. PEDOT-S derivativeshave been reported as having high conductivities from a few mS cm–1 up to 1089 S cm–1, a high aqueoussolubility of >40 mg/mL, and high biocompatibility.21 In addition,PEDOT-S has been synthesized by electropolymerization23 and oxidative polymerization; in a normal organic lab setting, thelatter is an attractive option because of its simplicity, low cost,and scalable synthesis.Bioelectronics, whichaims to modulate and monitor biological processes,is an evolving field complementing traditional small-molecule drugtreatments and other new modality strategies.23 and closelyrelated S-EDOT monomers20 into PEDOT-Sand S-PEDOT, respectively. These methods typically use Fe(III) orFe(II), either as a stoichiometric oxidant or in catalytic amountsin the presence of a persulfate as a stoichiometric oxidant. Differentsolvents, reactants, reaction times, temperatures, and workups havebeen reported. More recently, examples of direct C–H arylationpolymerization reactions targeting the synthesis of controlled copolymersof PEDOT analogues, as well as the PEDOT-S homopolymer, have beenreported.26As an emerging alternative conductivepolymer to PEDOT:PSS, therehave been several reported methods for the oxidative polymerizationof EDOT-S–1) in a model system.Although there are few publications on PEDOT-Scompared with thosefocusing on PEDOT:PSS, deciphering the properties  of the synthesized materials using differentmethods—including postprocessing, which is aimed at specificutilities—just by reading the literature is a challenge. Therefore,we decided to reproduce six original protocols that we found in theliterature at a lab scale  and compare them in a setting applicable forour use: highly soluble self-organizing nanoparticle dispersions basedon PEDOT-S forming a conductive hydrogel in a biologically relevantenvironment. We describe the discovery that some methods generatedPEDOT-S/-OH heteropolymers not PEDOT-S homopolymers. The importanceof this heterogenicity for solubility and self-aggregation was evaluatedand used to generate an injectable hydrogel displaying high conductivities were added to a reaction vial, that was capped andflushed with nitrogen. 1 M aq. H2SO4 (5 mL)was added, and nitrogen flushing continued during addition. (NH4)2S2O8  was dissolved in 1 mL of Milli-Q water, giving a clearsolution that was added dropwise using a syringe to the above solution,causing a color change: first to green and then to deep blue. Thereaction mixture was stirred at rt for 20 h under nitrogen. The mixtureturned viscous within 2 h after the addition of ammonium persulfate.After 20 h, the crude material was diluted with 10 mL of water. Cationexchange resin  was rinsed by shakingin Milli-Q water (2 × 20 mL). The resin was collected by filtrationand added to the reaction mixture, which was shaken overnight (shakingtable). The cation resin was then removed by filtration and washedwith water (3 × 10 mL); the collected deep blue aq. solutionwas poured into a glass vial containing anion exchange resin  previously rinsed as for the cation exchangeresin. The mixture was shaken overnight. The anion exchange resinwas then removed by filtration, washed with Milli-Q water (3 ×10 mL), and the resulting deep blue solution was freeze-dried, giving214 mg (71% yield) of the polymer as a dark blue solid.EDOT-S  and Fe(II)SOCyanine3 NHSester  was added to polyornithinehydrobromide  in 1 mL Ringer medium (pH7.25). After 1 h, the reaction mixture was spin-filtered (0.22 μm),and the final solution was used without further purification.PEDOT-S  and polyornithine-CY3  were injectedusing a 10 μL Hamilton syringe into an agarose gel (0.5% inRinger buffer or Milli-Q water) in 6-well microtiter plates with a15 mm separation (spot size 2 mm). After 2 h, the size of the spotwas measured again. After 15 days, the diffusion ratio was measuredas the distance between the PEDOT-S injection spot and the polyornithine-PEDOTprecipitation divided by the total distance between the PEDOT-S andpolyornithine-CY3 injections.A diffusion ratio for PEDOT-Sand polyornithine (CY3) of 0.5 would mean an equal diffusion ratio.A5 polymer solution  was addedinto isopropanol (20 mL) in a Petri dish, which resulted in a polymerfilm at the bottom surface. After 10 min, the film was lifted usinga tweezer upon which it spontaneously folded into a wire. The wirewas then dried for 1 h in air.The A5 wirefrom the previous step was dipped into a solution with 0.1 M CaCl2. The cross-linked A5 wire was then air-driedfor 2 h before analysis.The A5 wire, followed by cross-linking of the wire),75 μL (10 mg/mL) A5 and 25 μL (5 mg/mL) ironnanoparticles ,were mixed to form an A5-wire containing the magneticnanoparticles. The cross-linked wire was confirmed to be magneticusing a neodymium magnet.Using themethods above . Twenty laser shots were employed for each analysis witha laser energy set to 30 μJ. The MS were processed using Xcalibursoftware v3.0.63 (Thermo Scientific).Matrix-assistedlaser desorption ionization (MALDI) mass spectra (MS) were obtainedon an LTQ Orbitrap XL . PEDOT-Spolymers were dissolved in 0.1% TFA at a concentration of 0.2 mg/mL.The MALDI matrix 2, 5-dihydroxybenzoic (DHB) acid was prepared at10 mg/mL in 50:50 [v/v] methanol/0.1% TFA in water. The PEDOT-S solution(0.5 μL) was air-dried on the flat surface of a stainless steelplate. Next, 0.5 μL of DHB was deposited over the PEDOT layer,and the mixture was allowed to dry. Full mass scans were acquiredin positive mode in the Supporting Information.Gel permeation chromatography(GPC) was performed by PSS Polymer Standards Service GmbH. Detailsof running conditions, chromatograms, and molecular weight distributionsare given in the A5 wire in water and the PEDOT-Sin agarose were measured in a similar manner; however, in this case,current–voltage sweeps were performed at several electrodedistances. The transmission line model was used to extract the conductivityof the polymers.A Keithley 2612B connectedto Au-coated W electrodes  wasused to characterize the electrical properties. For the dried samples,a 3 μL droplet [10 mg/mL] of the PEDOT-S dissolved in Milli-Qwater was placed on a glass slide. After drying in air, the PEDOT-Swas contacted at an electrode distance of 2.5 mm. An applied biaswas swept between −0.5 and +0.5 V and the current was measured.Linear current–voltage relationships were typically recorded.The conductivities of 27 The areaof the A5 film was defined by peeling a sacrificial layerof parylene C by using a peel-off technique. For the four-point probemeasurements, the conductivity was calculated from the sheet resistanceI is the appliedcurrentat the outer probes and V is the measured voltageat the inner probes. d is the thickness of the films,and it was measured using a Dektak profilometer. The conductivitywas then g = 1/ρ (S cm–1).Similarly, the conductivity caused by using patterned Au lines wascalculated by the geometrical characteristics of the filmsw is the width and l is the length. The conductivitywas calculated using g = 1/ρ (S cm–1).The polymers were diluted in deionized waterat a final concentrationof 10 mg/mL, and the solution was sonicated for 1 h. The sonicatedsolution was spin-coated on top of either parylene-C coated glassslides or on patterned Au lines with a width of 2.5 mm, a length of15 μm, and a distance between each electrode of 15 μm.The fabrication of the electrodes was based on a previously reportedparylene fabrication process.A5 films,and an Ag/AgCl pellet was immersed in the PBS electrolyte.For organicelectrochemical transistor (OECT) measurements, phosphate bufferedsaline (PBS) was drop-casted on top of the With the aim of finding a PEDOT-S formulationsuitable for biomedicalapplications, we reproduced six previously reported synthetic methodsfor PEDOT-S by chemical oxidative polymerization of EDOT-S 1.23 Reynolds,21 Konradsson,19 andOkuzaki20) and an inventor in the caseof a patent method (Sautter28). For clarity,the only iron-free method is denoted as C–H arylation (Yu26). Furthermore, the original Okuzaki method producedPEDOT with butane-2-sulfonate, which is denoted S-PEDOT. For comparisonwith the other methods, we have used the Okuzaki method to synthesizePEDOT-S with butane-1-sulfonate, which we named A5 and A1 (5 and 1 stand for the percentage of the concentrationof the monomer in the reaction mixture) to see if there was a correlationin the method using the butane-1-sulfonate compared with butane-2-sulfonate,the one used by Okuzaki. In addition, the original S-PEDOT synthesiswas also reproduced.To facilitate the discussion, the methods are represented by thecorresponding author  as an oxidant,added to the reactionmixture as Fe(III) or Fe(II), either in a catalytic amount (Konradsson),in a 3–4 equiv excess  or 0.6equiv (Okuzaki). In addition, the Konradsson and Okuzaki methods usepersulfate as a stoichiometric oxidant. Except for the Reynolds method,which is performed in chloroform, all methods use water as the solvent1. DifferSupporting Information for synthetic details)yielded materials that resulted in dark blue solutions when dissolvedin deionized water. The palladium catalyzed method resulted in a materialthat gave a brown-colored solution. This could be because of the workupmethod used to purify the polymer, which only uses precipitation withacetone, which, in our experience, is not enough to remove the excesssalt used in this method. The initial characterization of the reproducedPEDOT-S materials included UV–vis absorption spectroscopy ,which is typically observed for doped PEDOT polymers.For the polymer to be injectable and form a structurethat is conductivein vivo, the balance between solubility and diffusion in vivo is key.To increase the likelihood of self-aggregation into a conductive structure,the nanoparticles need to have high aqueous solubility while demonstratinglimited diffusion, but still enough diffusion to be able to occupythe backtrack (column injection) of the capillary trace and to reachnearby cells for seamless infiltration.31  prepared with Milli-Q water or Ringer buffer, thelatter is a water solution of physiological salt concentrations mimickingbody fluids. Aqueous solutions of PEDOT-S were injected in a 0.5 wt% agarose gel cast in Milli-Q water. All PEDOT-S derivatives showedcomplete diffusion; after 2 h, the distinct pattern from the injectionsite had transformed into a gray cloud in the gel  showed intermediate performance. Second to last came a group comprisingKonradsson (reported conductivity of 12 S cm–1)and Sautter and Reynolds (reported conductivity of 0.2 mS cm–1). At the very last with the lowest conductivity came the C–Harylation method. Except for the Konradsson PEDOT-S, the ranking ofthe PEDOT-S conductivities followed the reported conductivities, iftaking the freedom to expect the same conductivity for A5 synthesized by the Okuzaki method as for the original S-PEDOT (1089S cm–1). However, our measurements did not includeany post-treatments that might have been used in the original procedures.In addition, to figure out the contribution of different reagentsin the Okuzaki method, different PEDOT-S were produced, iterativelyremoving one reactant at a time. In one case, removing the acid fromthe methodology produced a material (A5Y) with a conductivitythat was similar to those of A1 and Zotti. This methodresembles a later method published by Konradsson and Berggren (reportedconductivity of 30 S cm–1).32We found that the conductivity strongly dependedon the syntheticprocedure, which spanned more than 4 orders of magnitude 1e. All pA5 had the highestconductivityin the dry state and was the only polymer that rendered a conductivestructure when injected into agarose, thereby making it an excellentcandidate for syringe-injectable bioelectronics.To conclude, we found that A5), a four-point probe measurementof conductivity was carried out in a laboratory blinded to sampleidentity  gave a conductivity of 33S cm–1. The Zotti and Konradsson methods were alsoanalyzed using the same setup, mainly because of the discrepancy inthe rank order between our two-point measurements and published values.Here, the four-point probe measurements gave 3 S cm–1 for Zotti, which is between the 1 and 5 S cm–1 published, and for Konradsson 6 S cm–1, whichis still two times lower than what has been published. However, theseresults corroborate with the rank order of the published results,indicating that the Konradsson PEDOT-S forms better films in thissetting. For comparison, we also synthesized the S-PEDOT originallymade by Okuzaki and co-workers.20 Thispolymer has a high reported conductivity (1089 S cm–1). The monomer was synthesized using the same protocol as that usedfor EDOT-S with 2,4-butane sultone in place of 1,4-butane sultone.The S-PEDOT showed a conductivity of 30 S cm–1,which is basically the same as that of A5.Because theOkuzaki method has not previously been reported forthe synthesis of PEDOT-S , providing a stable performance in aqueous environments.The peak transconductance was found to be at Vg = −0.3 V with a value of 1.2 mS, which is relatively high for a polymer withoutadditional dopants.35 Having identifieda synthetic procedure for PEDOT-S that resulted in a polymer thatformed conductive structures in the agarose model, we turned our attentionto better understanding the reason behind the differences betweenthe various PEDOT-S preparations. An initial hypothesis was that thepolymer length was the reason for these differences. This was alsobrought forward by Okuzaki as an explanation of the high conductivityfound when using their methodology. Thus, a size comparison of thedifferent PEDOT-S materials was performed to search for a possiblecorrelation between the polymer length and diffusion, conductivity,or both. Size determination of PEDOT-S is typically carried out eitherby GPC, MALDI-MS, or both. GPC has been reported to overestimate thesize of thiophene-based polyelectrolytes similar to PEDOT-S becauseof an expansion of the polymer chain caused by counterion dissociationand the associated charge repulsion between the charges in the material.36 Furthermore, PSS is normally used as a referencefor size determination in GPC. The PSS backbone incorporates sp3-hybridized carbons, whereas the backbone of PEDOT-S has mainlysp2-hybridization; thus, PSS can adopt conformers similarto a yarn ball, while PEDOT-S has a different extended structure becauseof the rigid π-conjugated backbone and electrostatic repulsionbetween the alkoxysulfonate side chains. Furthermore, strong molecularaggregations also have an impact on the GPC analysis.37 With theselimitations in mind, high-resolution MALDI-MS can still provide acomparison within a series of polymers and was used in this studyto determine and compare the molecular size of PEDOT-S obtained usingdifferent methods. We found that except for the Konradsson methodology,all other iron-catalyzed methods, including the methodology variationsbased on the Okuzaki method, for example, A5Y (Supporting Information), gave essentially thesame size: an average polymer length of seven to eight monomers anda maximum length of 11 (Reynolds and Zotti) and 12 (Sautter and A5) monomers. Konradsson’s protocol gave polymers withaverage of five monomers and a maximum length up to nine monomer units(see the Supporting Information for thehigh-resolution MALDI-MS spectra). This corroborates the MALDI-MSanalysis reported by Zotti of six to eight monomer units on averageand polymers of up to 15 monomer units. For the palladium-catalyzedpolymerization, we were unable to detect any oligomers on MALDI-MS,indicating that the C–H method did not produce PEDOT-S; themethod was repeated several times with the same outcome. Contraryto this observation, the UV–vis spectrum indicated the presenceof doped oligomers . These polymers were sentto an external contract organization for GPC analysis using both UV(254 nm) and refractive index (RI) detectors. However, a large polymerdispersion index was seen, mainly because of Mw, in the order of 100,000 Da, which is an indication of aggregation.Surprisingly, scanning the literature, except for the Okuzaki S-PEDOTpublication, we were unable to find any raw data  in the literature onGPC of PEDOT-S. However, the unpublished results38 showed a chromatogram of Mw distribution using a RI detector that gave the GPC results of Mn = 28,000 and Mw = 123,000, which resemble our data. In our analysis, the Mn values looked reasonable: A5 , Konradsson , and Sautter . The GPC Mn for A5 is in the same ballparkas reported for Okuzaki’s S-PEDOT. A time dependence on theGPC results was also noticed, where Mn, Mw, or both, increased when the sampleswere left for 12 and 24 h, prior to analysis (see the Supporting Information). It is interesting thatwhen using a RI detector, the Mn valueswere doubled compared with UV (254 nm), while the Mw values were similar; thus, the RI detector seems tooverestimate low-molecular-weights (Supporting Information). In addition, Konradsson and Berggren analyzedPEDOT-S using GPC analysis (Mn = 1700and Mw = 5500), indicating an averagemonomer length of five EDOT-S units, which is somewhat shorter thanour analysis of A5Y. Dynamic light scattering measurementsperformed on A5 in a 1 mM NaCl solution showed the presenceof nanoparticles with a diameter of 81 ± 32 nm in size . These results further corroborate the hypothesis that A5 forms aggregates in solution.Ourintention was to compare MALDI-MS with GPC of a set of PEDOT-S(Supporting Information for the MALDI spectra), this could not explain the difference indiffusion and conductivity. However, using high-resolution MALDI-MS,heterogeneity was identified in most of the methods, except for theones using an excess FeCl3 (Reynolds and Sautter), withoutthe addition of any acid or persulfate (S6). A closeranalysis of the MS showed that the obtained polymers in , rather than PEDOT-S,are a PEDOT-S/OH copolymer with various degrees of EDOT-OH incorporation of S-PEDOT; however, S-PEDOT had the same diffusion characteristicsas A5 in Ringer cast agarose. Interestingly, the MALDI-MSanalysis showed an S-PEDOT length distribution similar to that ofother PEDOT-S; thus, the methodology did not give the longer polymersas previously presented in our hands. Thus, the same conditions leadto substantial formation of EDOT-OH monomeric units in PEDOT-S, butnot in S-PEDOT. One conceivable mechanism that could explain theseresults would be an intramolecular acid-catalyzed ether hydrolysis1H-NMR of the EDOT-S monomer (seethe Supporting Information), which wasused as precursor, did not reveal any EDOT-OH. Furthermore, the verysame batch of EDOT-S had been used in the FeCl3 methods(Reynolds and Sautter) without yielding a copolymer, suggesting thatEDOT-OH is formed in the polymerization reaction and does not resultfrom a pre-existing contamination   3e. AddinA5 PEDOT-S and S-PEDOT couldpartly be explained by the presence of sulfuric acid in the polymerization.A plausible explanation for the high conductivity is a combined effectof acidic doping (through sulfuric acid) and oxidative doping (throughammonium persulfate). In addition, the presence of EDOT-OH could generatenanocrystals that are doped during synthesis and that are not availablefor postdoping with acid. As previously described, we used the Okuzakiprotocol—but without the acid—for the synthesis of A5Y. The results looked similar in MALDI-MS analysis comparedwith A5 but gave a lower conductivity. Dropwise additionof sulfuric acid to A5Y postpolymerization did not increasethe conductivity; thus, to achieve higher conductivity, acid shouldbe present during synthesis.The high conductivity of A5 into a Ringer solutionto form a hydrogel. The hydrogelwas contacted using two Au electrodes, here biased at −0.5V, and the resulting current was registered. Although biased, we addedor removed Ca2+ ions from our medium and could detect acorresponding change in the current, where higher Ca2+ concentrationsresulted in larger currents (lower resistance) . Importantly, the increased conductivity couldnot be linked to geometrical changes of the A5 becauseonly modest swelling was seen when moving it to CaCl2 solutions. To further investigate theseswelling characteristics, A5 solution (10 mg/mL) wasadded to isopropanol, which led to the formation of wires (see the Supporting Information). Drying of the wet wireto a dry state resulted in a 25× reduction of the cross-sectionalarea . The addition ofRinger solution to the dry wire resulted in a 10× increase ofthe cross-sectional area forming a hydrogel, and with pure water,the structure was slowly dissolved. This shows that A5 form hydrogels with >90% water content and that ions are criticalfor maintaining the hydrogel structure.To study how the ions alter theelectrical properties, we injected A5, one batch was noticed as havinga more pronounced aggregation behavior, which could not be completelyexplained by the content of EDOT-OH monomers in the material. Wheninjected into Ringer solution, this batch (10 mg/mL) formed long threadsof an aggregated polymer that could not be reproduced using the 90/10PEDOT-S/-OHmaterial. The increased aggregation behavior of the one batch mightbe because of incomplete purification. In addition, ICP-AES analysisof the material showed that the A5 material containsiron, despite an extensive workup with both cation and anion exchangesin the synthesis of the material. The batch that had increased aggregationhad a higher content of iron, but there was only a modest difference(1.06 vs 1.03 mg/L (Fe at 238.204 nm)). Based on the average molecularweight of the polymers of approximately 2500–3000 Da, thesenumbers translate to approximately one Fe-ion (Fe(III) or Fe(II))per two chains. We then turned our attention to preloading the A5 with iron (Fe2(SO4)3)as a means to tune the aggregation properties. Mixing A5 (10 mg/mL) with 2 mM Fe2(SO4)3 orlower concentrations resulted in injectable solutions, but at 5 mM,precipitation occurred (2(SO4)3,the polymer solution was viscous and challenging to inject. At 1 mMFe2(SO4)3, the Fe-loaded A5 solution was still injectable and formed polymer threads when injectedinto the Ringer solution, which were stable over an extended timeperiod. We also synthesized A5 without Fe(II) in thepolymerization, relying only on persulfate as an oxidant. This reactionproceeded very slowly; therefore, the reaction time was extended to2 weeks (see the Supporting Information for details). The MALDI-MS measurements  showed a polymer of basically the same size as the othersbut with an increase in the EDOT-OH content. In the diffusion experiment,it was dissolved completely, and conductivity measurements showedlittle or no conductivity in the range of the C–H method. Interestingly,the GPC analysis showed Mn = 3200 and Mw = 18,000 with a reasonable DPI of 5–6(Supporting Information), which is indicativethat this material is not prone to aggregation as the others  and that incorporation of Fe ions is important inoxidative polymerization to obtain a conductive material. Similarresults were previously shown by Konradsson and co-workers.19 Adding Fe3+ to A5 postpolymerizationresulted in a slight reduction of the conductivity . These conductive hydrogels remainedstable in pure water over an extended time (>7 months), both intermsof their conductivity and structure. We sought to enable externalmotion control of this water-stable A5 to allow for thecreation of functional devices such as electrical switches. To realizethis, we mixed the A5  with 5nm iron nanoparticles , followed by extrusioninto isopropanol, which resulted in the formation of a several cmlong conductive polymer wire. The wire was moved to 0.1 M CaCl2 to ensure ionic cross-linking, hence making it water stable.We then moved the formed hydrogel to pure water when performing electricalmeasurements to minimize the conductivity of the surrounding medium.An interesting feature of A5/FeNP hydrogels were independentlycontacted using our two electrode setups (A5/FeNP) wires weremoved around. When in proximity, albeit without electrical contact,we added a permanent magnet underneath the Petri dish containing the A5 wires. The magnetic field from the magnet pulls the wirestogether or pushes them apart, depending on their relative locations.The external magnet induced a high enough magnetic force onto thenanoparticles to move the A5 wires into electrical contactin a reversible manner. For over 2 min, we moved the A5 wires into and out of contact five times (see movie in the Supporting Information). When the wires wereseen to move together, we observed a corresponding jump to contactin our current reading (A5 hydrogels by incorporating Fe3O4 nanoparticles during extrusion. The addition of 15wt % of 5 nm magnetic iron nanoparticles (Fe3O4) to A5 reduced the conductivity close to 100× that forms water-dispersiblenanoparticles that self-aggregateinto a highly conductive hydrogel (1–5 S cm–1) when injected into a physiologically relevant model. The conductivehydrogel was found to be water stable over many months, and we usedits properties to make devices such as magnetoelectric relays. Tothis end, most of the synthetic methods of PEDOT-S that have beenpublished were reproduced and analyzed using high-resolution MALDI-MS.Surprisingly, only the methods using excess FeCl3 as thesole oxidant resulted in the homopolymer PEDOT-S, whereas all theother methods gave a copolymer of P(EDOT-S/EDOT-OH) in different monomerratios. The formation of the copolymer dictates the aggregation behaviorof the PEDOT-S derivatives, which impacts its characteristics to alarge extent. Furthermore, all the methods gave polymers of the samelength, on average seven to eight monomers long, except for one publishedmethod by Konradsson, which gave shorter polymers with an averagelength of five to six monomers. Thus, three classes of PEDOT-S derivativeswith potentially different uses, short and long copolymers and homopolymers,were identified. In general, PEDOT-S derivatives are closer to thedefinition of oligomers than classical polymers, as also noted byZotti et al. Comparing the MALDI-MS analysis with GPC was difficult,the latter highly overestimates the length probably because that theseoligomers are highly prone to aggregation. For GPC analysis, it isalso important to consider the difference between using RI or UV detectors;the former overestimates the polymer weights in the lower range, impacting Mn, but not the higher ones represented by Mw. All the methods used in the polymerizationshow conductivity values in the same rank order as published in theoriginal publications using two-terminal and four-point probe measurements.One exception was Konradsson’s shorter polymer, which gavea lower conductivity using the two-point measurements, but fall intothe rank when a more elaborate film-forming procedure was used alongwith performing four-point probe characterization. Using no iron catalystin the reaction still gave a PEDOT-S polymer of the same length asthe others, but showed little to no conductivity and little to notendency to aggregate despite the formation of a copolymer. Incorporationof iron ions between the oligomers seems to be important. A5, together with the S-PEDOT published by Okuzaki, displayed the highestconductivities, both about 30 S cm–1. This is considerablylower than the published values (1089 S cm–1) forS-PEDOT, but no postprocessing optimization was performed. Copolymerswere synthesized and evaluated by adding additional amounts of EDOT-OHto EDOT-S in a copolymerization. This generated soluble dispersionof conductive materials up to the point of 20% additions, above whichparticles started to form, which was not deemed useful for our specificapplication. We are currently applying these materials, including A5, in in vivo studies of conductive materials.In conclusion, we haveprepared a PEDOT-S derivative (


Methionine is an aliphatic, sulfur-containing, essential amino acid that has been demonstrated to have crucial roles in metabolism, innate immunity, and activation of endogenous antioxidant enzymes, including methionine sulfoxide reductase A/B and the biosynthesis of glutathione to counteract oxidative stress. Still, methionine restriction avoids altered methionine/transmethylation metabolism, thus reducing DNA damage and possibly avoiding neurodegenerative processes. In this study, we wanted to study the preventive effects of methionine in counteracting 6-hydroxydopamine (6-OHDA)-induced injury. In particular, we analyzed the protective effects of the amino acid L-methionine in an in vitro model of Parkinson’s disease and dissected the underlying mechanisms compared to the known antioxidant taurine to gain insights into the potential of methionine treatment in slowing the progression of the disease by maintaining mitochondrial functionality. In addition, to ascribe the effects of methionine on mitochondria and oxidative stress, methionine sulfoxide was used in place of methionine. The data obtained suggested that an L-methionine-enriched diet could be beneficial during aging to protect neurons from oxidative imbalance and mitochondrial dysfunction, thus preventing the progression of neurodegenerative processes. Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder, with prevalence increasing with age. The molecular mechanism of PD is still undefined, but one important factor in the disease pathogenesis is oxidative stress ,2, whichHigh levels of reactive oxygen species (ROS) detected in PD are due to the mitochondrial impairment leading to protein oxidation and aggregation as well as apoptosis . UnfortuIndeed, nutraceuticals and antioxidants are presently receiving attention as adjuvants in PD treatment, and several investigations using both in vitro and animal models of this disease are published. The antioxidant dietary supplement can help to prevent extreme oxidative stress environments by limiting ROS production and counteracting ROS-related damage.β-synthase (CBS), cystathionine γ-lyse (CTH), glutamate-cysteine ligase (GCL), and glutathione synthase (GS) [α-synuclein over-expression [In particular, in this research we focused on the potential of methionine, which is an essential sulfur-containing amino acid , constitase (GS) . Methionase (GS)  reportedpression .Another amino acid that raised interest for its antioxidant potential is taurine. Taurine is a beta-amino acid with a sulfonate group and lacking carboxyl group. Diet is the main source of taurine, but it is also endogenously synthesized via different synthetic pathways starting from cysteine, homocysteine, and cysteamine . In the 2) [Based on this evidence, we studied the potential effects of the amino acid L-methionine on an in vitro model of PD comparing it to the known antioxidant taurine. In particular, we tried to dissect the underlying mechanisms to gain insights into the potential of methionine treatment in slowing the progression of the disease by maintaining mitochondrial functionality. In addition, to ascribe these effects to mitochondria and oxidative stress, especially for methionine, Met-O was used in place of methionine because in the presence of a strong oxidant it is further irreversibly oxidized to form methionine sulfone (Met-O2) .®, Corning, NY, USA) supplemented with glutamine and antibiotics. Differentiated SH-SY5Y cells are broadly accepted for in vitro studies requiring neuronal-like cells. Differentiated SH-SY5Y cells are low-cost to culture, and the ethical concerns related to primary human neuronal culture are avoided. Moreover, since SH-SY5Y cells are human-derived, they express several human-specific proteins and isoforms that would not be inherently present in rodent primary cultures [2 and after 24 h were grown in 10 μM Retinoic acid (RA)  with 1% FBS for 3 days. After 3 days, the media were replaced with fresh 12-O-tetradecanoylphorbol-13-acetate (TPA)  for another 3 days of differentiation. For the setup of the in vitro Parkinson’s disease model, the SH-SY5Y cells line was treated with 6-OHDA  (ranging from 25 µM to 100 µM) for 24 h after differentiation [The human neuroblastoma cell line SH-SY5Y (ATCC Cat. CRL-2266) was cultured in Dulbecco’s minimum essential medium (DMEM) , as previously described , and mea4 cells for well. After treatments, cells were incubated with the Alexa Fluor 488 Annexin V  in annexin binding buffer (Cat. V13241) for 15 min at 37 °C. After incubation, the multiwell was transferred to the IncuCyte controller (Sartorius) for 4 h. Data analysis was performed with the software IncuCyte S3 2018 and based on the green area/phase area’s fluorescence intensity.The apoptosis assay was performed with the IncuCyte Live System ; cells were seeded on a multiwell plate at a density of 1.5 × 10g RPM for 30 min at 4 °C. Protein concentration was assayed by the micro-BCA kit  according to Brandolini et al. [The cells were washed with cold 1× phosphate-buffered saline buffer followed by detachment with scrapers. Pellets were collected and resuspended in cell lysis buffer containing protease and phosphatase inhibitor. After, the lysate was collected and maintained on ice for 30 min. Finally, the total protein was extracted by centrifugation at 14,000× i et al. .SDS-polyacrylamide gel electrophoresis was performed, running samples (15–30 µg/lane) on 8–15% polyacrylamide denaturing gels. Proteins were transferred to polyvinylidene difluoride membranes and then blocked in skimmed milk for 1 h. The membranes were then incubated with: anti-cleaved caspase 9 ; anti-p-JNK ; anti-p-AKT; anti-PI3K; anti-p-Bcl2 ; rabbit monoclonal anti-p-Creb ; anti-mBDNF ; anti-p-TrkB ; anti-p-Nrf2 ; anti-Mn-Sod2 ; anti-Catalase ; anti-Gpx ; anti-GstP1 ; anti-HNE ; anti-Sirt1 ; anti-p-Foxo3a ; anti-Msrb2 , anti-Drp-1 , anti-Opa-1 , and anti-Mfn1/2  at 4 °C overnight. After three washes (5 min each) in TBS-T, they were further incubated with 1:20,000 horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG. According to the manufacturer’s instructions, the protein bands were visualized by ECL Prime  Western blotting detection reagent. Alliance Q9 MINI WL  was used to image chemiluminescent bands. To perform the analysis of each band intensity the NIH ImageJ program was used. Anti-β-actin   or GAPDH  was used as the loading control depending on the molecular weight of the proteins assayed. The relative peak intensity of each marker band analyzed was normalized with respect to the loading control band.Protein carbonyls were analyzed by Western blot analysis according to the manufacturer’s protocols . A total of 30 μg protein from protein lysate was mixed with 2,4 dinitrophenylhydrazine and blotted using a primary antibody specific to dinitrophenylhydrazone-derivatized residues (OxyBlot™) and a secondary antibody against the first one (OxyBlot). Protein carbonyls were visualized by Alliance Q9 MINI WL and revealed using enhanced chemiluminescence and quantified by densitometry using ImageJ software. The bands obtained were normalized by Coomassie blue staining of the total gel.4. After treatments, the cells were washed three times with PBS. Then, the cells were incubated with Mitotracker deep Red  in HBSS buffer at concentration of 1 µM for 15 min. Then, cells on coverslips were washed three times in PBS and then fixed with 3.7% formaldehyde in PBS for 15 min at RT. After further washes in PBS, coverslips were mounted with Vectashield mounting medium containing DAPI nuclear dye. The observation was performed with a confocal microscope Leica TCS SP5, and images were acquired with LCS Leica confocal software SP5 . Quantitative determination of mitochondrial fragmentation was performed as previously described [MitoTracker is a fluorescent dye that stains mitochondria in live cells, and its accumulation is dependent upon membrane potential. In particular, cells were seeded on the coverslips at a density of 1.5 × 10escribed . Mitochoescribed .Subsequently, cells were stained with Mitosox Red reagent, which is a novel fluorogenic dye specifically targeted to mitochondria in live cells. Mitosox Red reagent permeates live cells where it selectively targets mitochondria. It is rapidly oxidized by superoxide but not by other reactive oxygen species (ROS) and reactive nitrogen species (RNS). When the red reagent is oxidized by superoxide, it exhibits red fluorescence. Cells were marked with Mitotracker Green FM, washed three times with PBS, and then incubated with MitoSox  in HBSS buffer at 1 µM concentration for 10 min. After different washes in PBS, coverslips were mounted with Vectashield mounting medium containing DAPI nuclear dye. The observation was performed with a confocal microscope Leica TCS SP5, and images were acquired with LCS Leica confocal software SP5 . The data analyses were performed using NHI ImageJ. The fluorescence intensity for Mitotracker green was normalized on the nuclei (DAPI), while the fluorescence intensity for Mitosox Red was normalized on the normalized Mitotracker intensity.4 cells per well (optimized to have a better staining). After treatment, the cells were incubated with TMRM reagent  in PBS for 15 min at 37 °C. After incubation, the multiwell was transferred to the IncuCyte controller for 24 h.Healthy mitochondrial membranes maintain a difference in electrical potential between the interior and exterior of the organelle, referred to as a membrane potential. Tetramethylrhodamine methyl ester (TMRM) is a cell-permeant dye that accumulates in active mitochondria with intact membrane potentials. If the cells are healthy and have functioning mitochondria, the signal is bright. Upon loss of the mitochondrial membrane potential, TMRM accumulation ends, and the signal fades or disappears. The TMRM assay was performed with the IncuCyte Live System. The cells were seeded on the multiwell plate at a density of 2 × 104 cells/well  and grown overnight in DMEM 10% of FBS, then differentiated as described above. After complete differentiation, cells were treated as described above. On the day before the Seahorse assay, the cartridge was hydrated and incubated overnight at 37 °C in the absence of CO2. On the day of the assay, cell medium was replaced with unbuffered DMEM  supplemented with 5 mM glucose and 1 mM sodium pyruvate , and incubated for 1 h at 37 °C in the absence of CO2. Medium and reagents were adjusted to pH 7.4 on the day of the assay. After four baseline measurements for the oxygen consumption ratio, cells were sequentially challenged with injections of Mito Stress drugs prepared following the manufacturer’s instructions. The final concentrations used for each drug were 1 μM oligomycin (ATP synthase inhibitor), 1 μM FCCP , and 0.5 μM rotenone/antimycin (complex I and III inhibitors). For the normalization in port D, Hoechst 33,342 solution was injected, and at the end of the run, the plate was read using a microplate reader . The data and graphs generated at the end of the Mito Stress assay were extracted using Wave software.The Seahorse XF96e Extracellular Flux Analyzer  was used to generate the bioenergetic profiles of differentiated neuroblastoma SH-SY5Y cell line upon treatments. Live-cell analyses of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Mito Stress test (Agilent). Cells were cultured on a Seahorse XF96 cell culture plate  at a density of 5 × 10The total SOD activity was measured by an enzymatic method using an SOD assay kit purchased from Abcam, UK. SOD activity was assessed by measuring the rate of reduction of WST-1 , which produces a water-soluble formazan dye upon reduction with a superoxide anion. The absorbance of WST-1 was measured at 450 nm. The total measured SOD activity was calculated using the decrease in color development at 450 nm compared to the control.https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf, accessed on 5 June 2021), and differential expression analysis was performed. DESeq2 is a bioconducter package implemented in R . Aggregated quality control and trimming to remove various types of unwanted sequences  were performed with MULTIQC and Cutadapt tools, respectively . Clustering and functional analysis of differential expression genes was performed by CHIMERA (Metrica e cluster construction). Interactors’ network was built with STRING .Cells were cultured as described, and RNA was extracted with Trizol  following the manufacturer’s instructions. Data were obtained from SH-SY5Y untreated cells as a control and cell treated either with 6-OHDA alone or in combination with Met, Met-O, or Tau. All the experiments were performed in triplicate. RNA-seq experiments were performed at Lexogen, a biotech company expert in expression profiling technologies using the QuantSeq 3′ mRNA-Seq Library Prep Kit, which is a library preparation protocol designed to generate libraries of sequences close to the 3′ end of polyadenylated RNA. Data were aligned by the STAR tool  v6.8 was used to perform Gene Ontology (GO) enrichment analysis . We perf-Network edges represent data from high-throughput lab experiments, (conserved) co-expression, and previous knowledge in databases.-The interaction score cutoff used was 0.7 (high confidence).-The list of proteins used was generated starting from the expression data achieved by RNA-seq. The log(2) of the 6-OHDA+drug (methionine or taurine) compared to 6-OHDA alone was calculated as the difference between log(2) observed in the 6-OHDA+drug and 6-OHDA samples compared to untreated cells, respectively. For the analysis we used the genes with a log(2) >of 0.4 and <−0.4; these genes are mentioned in the text as “recovered”.STRING is a database of known and predicted protein–protein interaction (PPIs), in which interactions are derived from five main sources: genomic context predictions, high-throughput lab experiments, (conserved) co-expression, automated text mining, and previous knowledge in databases . For thip < 0.05.Prior to the analysis, the Shapiro–Wilk test was used to assess the normal distribution of the samples. Data are mean ± SE of 3 different biological experiments. Statistical analysis was performed by one-way ANOVA following Tukey’s post hoc test. For grouped analyses (TMRM), two-way ANOVA was used followed by Tukey’s post hoc test. The level of significance was set at In a preliminary series of experiments, cell viability dose–response curves were performed for 6-OHDA, Met, taurine, and Met-O, to determine the best concentration to be used in subsequent experiments . On thisAs illustrated in p < 0.05). Interestingly, cluster analysis revealed that the general pattern of gene regulation afforded by 6-OHDA is reverted by Met and taurine addition, while Met-O, as expected, was not able to recover the general pattern of gene expression dysregulated by 6-OHDA  around the Msr proteins   . TogetheWe, then, decided to explore by bioinformatics analysis the functional enrichment of the list of genes dysregulated by 6-OHDA treatment and recovered by Met or taurine. The analysis was performed by STRING database. STRING generates PPI networks, confirming the enrichment for GO terms and protein interactions associated with apoptosis/cell death and neurogenesis mainly among the transcripts respectively up-or down-regulated by 6-OHDA and recovered by the treatments, as reported in All together, these results suggested that Met could exert protective effects in an in vitro model of PD, possibly protecting neurons. We then performed further experiments to validate this hypothesis.Apoptosis evaluation by Annexin V live assay by IncuCyte system and Western blotting (WB) analysis for cleaved-caspase 9 and p-JNK are reported in The survival pathway  and the neuroprotective pathway  assayed in control and treated cells are shown in In In Mitochondrial functionality was assayed in all the conditions tested . In partTMRM was used to test the integrity of mitochondrial membrane potential, which is accumulated in active mitochondria . 6-OHDA Healthy mitochondrial morphology and dynamics are essential for maintaining mitochondrial function; for this reason, Mitotracker deep Red was used to evaluate mitochondrial fragmentation. The mitochondrial morphology of our cell model  showed tubular networks, while upon 6-OHDA challenge, the mitochondria were mainly shorter and smaller size, suggesting mitochondrial fragmentation. Notably, mitochondrial morphological changes were attenuated by Met and taurine treatments .Mitochondrial morphology is likely to be the result of the interplay between mitochondrial division and fusion. Mitochondrial fusion is controlled by Mfn1/2 and Opa-1, whereas mitochondrial fission is mainly controlled by Drp-1. To study whether mitochondrial fission and fusion were influenced by 6-OHDA and the tested compounds, the expression of these factors was assayed by Western blot . We founIn line with previous results, Met-O presence was ineffective in producing the protecting effects observed for Met on mitochondrial functionality .We then investigated the mitochondrial functionality in our cell model using Mito Stress assay by Seahorse Extracellular Flux Analyzer to characterize the cells’ bioenergetics profile upon treatments . In partWith the injection of oligomycin, cells treated with 6-OHDA and Met as well as taurine maintained much higher oxygen consumption than 6-OHDA alone or Met-O, leading to significant differences in ATP production through mitochondrial respiration and proton leak B. InductDopaminergic neurons are already damaged at the time of diagnosis of PD and the first appearance of symptoms. Therefore, the interest in protective treatments aimed at decreasing oxidative stress and delaying disease progression is very high. In this regard, it is worth considering that oxidative stress, mitochondrial dysfunction, and redox balance perturbation are observed in neurodegenerative diseases, including PD ,39,40. RIn addition, Wang et al., 2019 , demonstR-sulfoxide has been so far detected [In agreement we demonstrate herein that the Msr system, in particular mitochondrial MrsB2, plays a pivotal role in cell rescue and mitochondria viability upon 6-OHDA in the presence of methionine, thus suggesting that 6-OHDA determined, in this cellular model, an increase in R sulfoxide diastereomer , since ndetected . RNAseq detected , which iBased on these results, it seems conceivable that an L-methionine-enriched diet could show therapeutic potential for human aging and age-related disorders, protecting neurons from oxidative imbalance and maintaining mitochondrial functionality. In


E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in  Due to many scientific advances, with the use of modern molecular biology tools, the biopharmaceutical industry has become a multibillion-dollar industry. Through the last years, the biotechnology industry invested massively in the production of these molecules indicating a substantial growth dynamic in the future. The “Global Protein Therapeutics Market Outlook 2020” report estimates that by the end of 2020, this market may reach $208 billion dollars .Biopharmaceuticals are characterized as molecules with therapeutic proprieties, produced through biotechnological processes. These drugs are currently used in a wide array of medical specialties and sometimes are considered the most effective treatments. Therapeutic proteins are used in a broad range of diseases, such as cancer, metabolic disorders and immunotherapy .Escherichia coli. The E. coli expression system is very attractive for industrial processes as E. coli cell molecular biology, uncomplicated manipulation, and easy scale-up are broadly known, favoring target protein production [E. coli expression host could be considered the best choice for the biotechnology industry, the expression of heterologous proteins still presents many limitations: inadequate disulfide bonds, the presence of endotoxins, chaperone absence, codon incompatibility, and protein aggregation in inclusion bodies. It is estimated that E. coli is capable of expressing 75% of human proteins; however, only 25% of these molecules are produced in a soluble and active structure [Several biopharmaceuticals are produced in prokaryotic systems, such as the bacteria oduction ,4. Even tructure ,7. As prE. coli can be increased by fusing molecules (solubility tags) with the target protein. Solubility tags are extremely soluble proteins, or fragments of these proteins, that can also exert chaperone activity. When fused to the protein of interest, they tend to improve folding and protein solubility [Production of soluble proteins in lubility . Severallubility ,11,12,13lubility . Fh8 andFasciola hepatica that was initially proposed to be used as a diagnostic tool and vaccine and drug development against the parasite [Cryptosporidium parvum protein (CP12), human interleukin-5 (IL-5) and Toxoplasma gondii oocyst wall (TgOWP). These studies reported an increase of 3 to 16 times in production yield when compared to the same proteins without the associated tag [Fh8 (GenBank ID: AF213970.1) is one of the promising tags whose advantage is to provide effective solubility ,15. The E. coli is mediated by several Dsb family proteins, such as DsbA, DsbB, DsbC, DsbD, DsbE, and DsbG, which assist protein folding [98-Gly-Tyr-Cys101- portion. This amino acid arrangement makes DsbC highly reactive to the disulfide bond, thus assisting protein folding [E. coli is widely used to produce recombinant proteins, this expression host is not ideal for disulphide bond formation as E. coli cytoplasm exhibit a notably reducing environment favoring protein aggregation [The formation of disulfide bonds in  folding ,18. DsbC folding  and work folding . Even thregation ; thus, Dregation . For exaregation .A non-natural interferon, also called recombinant interferon-consensus, IFN-con, cIFN or rhIFN-con, was designed following the analysis of the amino acid sequences present in several subtypes of IFN-α . The antArboviruses (arthropod-borne viruses) represent a group of viruses that are transmitted by arthropod vectors . Mayaro Coronaviridae family and Betacoronavirus genus [SARS-CoV-2 belong to the us genus . Coronavus genus . The firus genus . Even thus genus , fever, us genus , pulmonaus genus . Interesus genus .E. coli fused with Fh8 (HF-IFN) or DsbC (HD-IFN) solubility tags and a six-histidine tag (His-tag) at N-terminus of the molecule or exclusively with N-terminal His-tag (H-IFN) and no solubility tag. The HF-IFN recombinant molecule was the best candidate for cIFN production among all analyzed constructs. The HF-IFN protein was produced in the soluble fraction of bacteria when cultivated in autoinduction medium at 30 °C. Additionally, the purified cIFN molecule was tested for antiviral activity against Zika, Chikungunya, Mayaro and SARS-CoV-2 viruses in vitro infection model. The data showed, for the first time, that the cIFN was successfully produced as a soluble molecule and showed strong antiviral activity.In this work, cIFN was produced in cifn gene was based on the article published by Mohammed et al. [cifn gene. Nco I and Xho I restriction sites were also included flanking regions of interest to allow cloning into pET28a(+) expression vector . The Bam HI site was incorporated into the 5’ end of the cifn gene and 3’ end of the fh8 or dsbc gene sequence. This was accomplished by introducing C671A, A670T, G669C silent mutations in the TEV protease cleavage site. The translation of 6xhis-fh8-cifn (753 bp) and 6xhis-dsbc-cifn (1194 bp) gene sequences resulted in the production of HF-IFN and HD-IFN molecules, respectively, and the translation of 6xhis-cifn (522bp) in the production of H-IFN   , 2.5 U TE. coli DH5α competent bacteria were used for plasmid amplification and molecular cloning. E. coli BL21(DE3) was used for gene expression. Both bacterial lineages were obtained from Invitrogen Corporation .Different culture media were used to evaluate cell growth and protein synthesis. Luria-Broth (LB) : cultureE. coli BL21(DE3) transformed with plasmid pET28a-hf-ifn or pET28a-h-ifn were cultured at 30 °C in Erlenmeyer flasks (1 L) with 200 mL of LB medium and induced with 0.1 mM IPTG when OD = 0.6. Samples from soluble and insoluble fractions were analyzed before and up to 6 h after IPTG induction.To compare the solubility with and without Fh8 tag, E. coli BL21(DE3) transformed with plasmid pET28a-hf-ifn was cultured at 25 °C or 30 °C in TunAir™ 300 mL-flasks containing 100 mL of medium and induced with 0.1 mM IPTG when OD600nm = 0.6 in HDF medium or lactose when glucose was exhausted in SDAB and autoinduction media. Samples of soluble fractions were analyzed before and up to 10 h after IPTG induction for HDF medium, and after glucose consumption for autoinduction and SDAB media, respectively, 4 to 5 h and 7 to 10 h after the beginning of cultivation. The same procedure was employed to evaluate HD-IFN production and solubility.To evaluate HF-IFN production and solubility, g for 5 min . The pellets were resuspended in 500 μL of saline and samples were sonicated for 2 min with pulses of 15 s on/off by the Branson Ultrasonic Sonifier S-250D . After sonication, samples were centrifuged at 15,000× g for 30 min and the soluble and insoluble fractions were separated. The pellet was resuspended in 80 μL of MilliQ water and 20 μL of SDS-PAGE 5× sample buffer. To analyze the soluble fraction, 80 μL of the supernatant was mixed with 20 μL of the 5× sample buffer. Samples were loaded into 15% polyacrylamide gel after heating samples at 96 °C for 5 min. The gels were stained with Coomassie Blue R-250 . Protein synthesis was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 15% gels. Samples with a volume equivalent to optical density (OD600nm) equal to 5.0 were withdrawn from the cultures and centrifuged at 15,000× g for 30 min and the supernatant was discarded. The pellet was resuspended in lysis buffer . Bacterial cells were disrupted by high-pressure homogenizer  through three passages at 1200 bar, keeping the cell suspension in an ice bath during the passages. All purification methods were conducted using 15 to 20 g of bacterial biomass.The bacterial culture was centrifuged at 5000× 2+ and chromatography was performed on ÄKTA Avant . The column was previously equilibrated with a buffer containing 20 mM phosphate, pH 7.4, 500 mM NaCl buffer and 10 mM imidazole. Elution was performed with increasing concentrations of imidazole .IMAC: The chromatography column (XK26) was packed with 50 mL IMAC-Sepharose 6 Fast Flow resin , the resin was charged with NiQ-Sepharose: The chromatography column (XK26) was packed with 74 mL of Q-Sepharose Fast Flow . The column was previously equilibrated with 50 mM Tris-HCl, pH 8.0. Elution was performed with increasing concentrations of NaCl (0 to 0.7 M).The fusion proteins encompass a TEV protease hydrolysis site, which is located between C-terminal end of the Fh8 or DsbC and N-terminal end of the consensus interferon. Thus, the TEV protease hydrolysis generates two protein fragments. The first fragment refers to the Fh8 or DsbC tag associated with the N-terminal histidine tag, which have molecular mass of 9.4 kDa and 24.2 kDa, respectively. The second protein fragment, containing the biopharmaceutical product of interest cIFN, has a theoretical molecular mass of 19.4 kDa .After Q-Sepharose and IMAC, HF- and HD-IFN samples were dialyzed against 1 L of phosphate buffered saline (PBS), PBS was changed three times every 1 h for removal of the imidazole. Then, the solubility tags were removed by hydrolysis with TEV protease . The hydrolysis reaction was conducted overnight using a 1:100 ratio (enzyme mass: protein mass) in 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 14 mM β-mercaptoethanol. In order to remove the hydrolyzed products, a HiTrap IMAC Sepharose FF column  was used. The column was equilibrated with 25 mM Tris-HCl pH 7.4 with 500 mM NaCl and 10 mM imidazole. The cleaved cIFN fraction was collected in the non-adsorbed fraction. Fh8 and DsbC solubility tags and TEV protease, which have a 6xHis tag in their N-terminal portion, as well as non-hydrolyzed product, were eluted with 500 mM imidazole.Protein purity was quantified by densitometry of SDS-PAGE bands using the BioRad GS-800 densitometer and Quantity One 4.6.3 software . The relative percentage of the target protein band was calculated by Equation (1):Total protein concentration was measured by the DC Protein Assay Lowry method  and the concentration of the target protein was calculated using the values of relative quantity according to the Equation (2):Purity determination of cIFN was performed by Agilent 1260 Infinity equipment . Sample and mobile phase were filtered at 0.22 µm. The TSKGel pre-column (PW-Type) and the HPLC column TSKGel G3000 PWXL  were used. The mobile phase is composed of 10 mM sodium phosphate, pH 7.5 and 150 mM NaCl. The flow rate used was 0.4 mL/min with a final injection volume of 20 µL. The protein peaks were detected by UV at 280 nm.2 atmosphere in all the different steps of the antiviral test. Zika virus  was kindly provided by Dr Pedro Vasconcelos (Evandro Chagas Institute), Mayaro virus  was kindly provided by Dr Maurício Nogueira (São José do Rio Preto School of Medicine), Chikungunya virus (CHIKV) was isolated from a serum sample taken during an outbreak in Sergipe, Brazil, 2017, which was kindly provided by Dr Alessandra Schanoski  and SARS-CoV-2 (SARS-Cov2/SP02/2020HIAE) was isolated from a nasopharyngeal swab and kindly provided by Dr Edison Luis Durigon .Vero cells  were cultured in D-MEM  with 10 % Fetal Calf Serum  and incubated at 37 °C with 5% CO4 cels/well) and grown for 48 hours until they reached 80% confluence. A sample corresponding to the cells of three wells was obtained after trypsinization and used for cell counting. Culture medium was replaced by serially diluted cIFN in new culture medium, starting at 640 pg/mL (SARS-CoV-2), 160 pg/mL (for CHIKV and MAYV) or 2.67 pg/mL (for ZIKV), and cells were incubated for two hours. Cells were then infected with 0.1 MOI of one of the arboviruses in study or 0.02 MOI for SARS-CoV-2. Controls (not infected cells or infected cells) were used for their respective viruses. Plates were incubated and a cytopathic effect was observed after 48 h for MAYV, 72 h for CHIKV and SARS-CoV-2, and 96 h for ZIKV. Following this, the culture medium was removed, plates were washed with PBS (pH 7.2), and staining was performed with NBB Solution  for 30 min. The staining solution was removed, the plates were rinsed with water and the absorbance was measured at a wavelength of 450 nm in a microplate reader . Mean absorbance (abs) values of each cIFN dilution, and infected and non-infected control analytical triplicates were used for the calculation of the infection inhibition rate for each experiment, defined by the Equation (3): Each antiviral test was performed in three different experiments with three analytical triplicates each. Firstly, cells were seeded in 96-well plates . The three EC50 values obtained for each virus tested were expressed as mean ± standard deviation. The cIFN used in this assay was purified from the HF-IFN construction.cifn and fh8 gene sequences were synthetized fused with the 6xhis-tag sequence for generating the hf-ifn insert. This construct served as template for a PCR reaction in order to amplify the cifn gene and to clone it to create the 6xhis-cifn (h-ifn) insert, i.e., the construct without the solubility tag. The dsbC gene sequence was synthesized and amplified by PCR. The dsbC insert was cloned into Nco I and Bam HI sites of pET28a-hf-ifn. This procedure allowed for the removal of the fh8 cassette and insertion of dsbC gene and resulted in the 6xhis-dsbC-cifn (hd-ifn) construct. The resulting plasmid was named pET28a-hd-ifn.Three variants of the cIFN fused to 6xHis, 6xHis-Fh8 or 6xHis-DsbC tags were constructed . The cifFigures related to the amplification, digestion and visualization of bands of interest are presented in In order to compare the solubility in the presence or absence of the Fh8 tag, two protein constructs were used: HF-IFN, which has the Fh8 tag fused to cIFN and H-IFN, which is not embedded with the solubility tag .E. coli.The evaluation of the results of gene expression shows that in the absence of Fh8, cIFN tends to aggregate and form inclusion bodies. H-IFN was detected in the soluble fraction only in the sample harvested 2 h after IPTG induction A and accThe relative amount of target protein was 20% for the two constructs analyzed after 2 h of IPTG induction . It is iE. coli for producing HF-IFN  and a complex medium (autoinduction medium). The results demonstrated that HDF and autoinduction media were superior when compared to the SDAB culture medium. The culture of g HF-IFN  reached g HF-IFN D. After g HF-IFN B, a 2.2-E. coli culture containing the pET28a-hd-ifn plasmid reached maximal production of the target protein in HDF medium at 25 °C after 11 h of induction with 0.1 mM IPTG  was evaluated in infection experiments using Vero cells, Zika, Mayaro, Chikungunya and SARS-CoV-2 viruses. The ECRS-CoV-2 . In prevE. coli bacterium was the pioneer expression platform in the production of biopharmaceuticals and was consolidated in industrial manufacturing. E. coli is widely used due to its high growth speed, low complexity in genetic manipulations, and high yield in the synthesis of recombinant proteins  [E. coli, such as: human insulin, interferon-α2a, anakinra, somatropin, among many others [The roteins) ,46. Curry others .E. coli.Among the existing variants of interferons, there is an artificial molecule called consensus. This variant has the characteristic of having the most prevalent amino acid sequence found in 14 subtypes of human interferons. Despite demonstrating higher biological activity than endogenous IFN-α, the production of cIFN remains a challenge . ResearcE. coli, different tags must be assessed individually as these molecules may behave differently according to the proprieties of the target protein [A common approach to overcome inclusion body formations is the use of solubility tags. This strategy may not only increase soluble protein recovery but may also aid in several purification methods ,49. Taki protein . A wide diversity of solubility tags is commercially available: maltose-binding protein (MBP), N-utilization substance (NusA), thioredoxin (TrxA), glutathione S-transferase (GST) and small ubiquitin-related modifier (SUMO) . Even thThe solubility tags used in this study were placed on the N-terminal portion of the target protein. The insertion of tags in the N-terminal portion is recommended, as it becomes the first portion to be translated. This strategy has the main objective to aid in the correct folding of the molecule of interest. In this way, tagged proteins in the N-terminal region were shown to have better folding when compared to cloning using tags in the C-terminal region . Comparicifn gene fused to the Fh8 tag (HF-IFN) not only promoted greater protein solubility, but also served to maintain the protein in the soluble fraction for a longer time during E. coli cultivation than the protein without solubility tag (H-IFN), which aggregated into bacterial inclusion bodies. These findings suggest that Fh8 could be a proper fusion tag for scaling up the bioprocess, since the downstream processing is also facilitated when there is less tendency of protein precipitation. To date, there are no reports in the literature relating recombinant cIFN synthesis associated with Fh8 in the E. coli expression platform.Our results are in agreement with the literature data that showed that Fh8 was able to increase solubility of several proteins, being as effective as the well-known MBP, NusA and Trx tags  and SUMOE. coli platform. It is suggested that each protein should be individually tested with different tags for better performance evaluation [DsbC is another solubility tag that was reported as being especially effective for proteins with disulfide bonds ,54. Herealuation .E. coli cultures. Furthermore, while DsbC represents 52% of HD-IFN molecule, Fh8 represents only 28% of HF-IFN. Thus, the production of 1 g of HF-IFN represents 0.72 g cIFN, while 1.0 g of HD-IFN contains 0.48 g of cIFN. SUMO has an intermediate size, 11.5 kDa, and represents 36% of SUMO-cIFN, as previously reported [The size of the tag must be carefully considered as a sizable tag, comprising a wide range of amino acids, may cause metabolic overburden and not promote protein solubility . DsbC isreported .Besides the use of solubility tags, it is also known that higher temperatures favor rapid bacterial growth, resulting in a higher production rate of the target protein and possibly in protein aggregation and the formation of inclusion bodies. Furthermore, the presence of strong promoters, such as the T7 promoter, high concentrations of IPTG, absence of chaperones and hydrophobic regions of proteins are complementary factors for the formation of inclusion bodies ,57.The decrease of the culture temperature from 37 °C to 30 °C as well as the concentration of IPTG in the culture medium from 1 mM to 0.1 mM favored HF-IFN production in the soluble fraction. These results are in agreement with literature data that reported an increase of recombinant IFN-α solubility by decreasing the temperature and reducing the IPTG concentration . This fiThe synthesis of HF-IFN and HD-IFN was also evaluated in three culture media: autoinduction, HDF and SDAB. These three media are suitable for good manufacturing practices (GMP), while LB medium is not, due to tryptone presence , and it The autoinduction culture medium  suppliesE. coli, recombinant IFN-α is also produced as inclusion bodies, refolded, and recovered. Many authors related their experience in purifying IFN-α from inclusion bodies mainly using Q-Sepharose [E. coli BL21-CodonPlus(DE3), which is a lineage of E. coli different from that used in this work. The author reports a recovery yield of 270 mg/L with a single-step DEAE-Sepharose chromatography. This result seems promising; however, we were not able to achieve the same results as reported by those authors without a solubility tag. In addition, Peciak et al. expressed soluble cIFN fused with solubility SUMO tag [E. coli SHuffle® suggesting that E. coli lineage may be crucial in order to obtain soluble cIFN. We also highlight the need for several chromatographic steps to exclude and discard contaminants that are together with cIFN in this process. These results indicate a favorable bioprocess for cIFN production as soluble protein, yet the production and purification processes must be polished.Even though there is no massive data about cIFN purification from epharose ,72,73 anepharose ,74. Whilepharose  when proSUMO tag  with a yThe functional characteristics of IFN-α are widely studied; however, the use of cIFN, a non-natural synthetic recombinant interferon, has not been described in the context of ZIKV, MAYV, CHIKV and SARS-CoV-2 virus until now ,75. Our E. coli BL21(DE3) cultivation for production of HF-IFN in autoinduction medium at 30 °C presented a final recovery of soluble cIFN of 8 mg/L, 5.5-fold higher when compared to HD-IFN. Thus, the production of cIFN fused to the Fh8 tag, in an autoinduction medium at 30 °C, is proposed as a viable approach to obtain this molecule in the soluble fraction. Even though we recovered low amounts of target protein, the aim to produce soluble cIFN could be an excellent alternative to inclusion bodies and should be further explored.The production of cIFN fused to the Fh8 tag (HF-IFN) favored protein solubility when compared to the H- and HD-IFN fusion molecules. Furthermore, Most important, our results demonstrate, for the first time, that the cIFN produced was able to inhibit the cytopathic effect of the ZIKV, MAYV, CHIKV and SARS-CoV-2 virus in VERO cells. Of note, this is a class of molecules known and approved by regulatory agencies such as the Food and Drug Administration (FDA), a fact that would accelerate a possible insertion of the molecule in a clinical setting in cases of public health emergencies, when the risks associated with IFN side-effects compensate the risks related to the disease ,77.